The proposed program seeks to develop and perfect combined on-line electrospray-liquid chromatography/mass spectrometry (ES-LC/Ms) for the sensitive and specific detemination of opioid peptides in biological matrices. The wide range of molecular weights of the known opioid peptides, the extremely low concentrations of these peptides in complex biological materials, and the ever-increasing number of individual peptides challenge current analytical methodology. This program will address these problems by investigating electrospray (ES) ionization which has been demonstrated to detect peptides at the 1-5 fM range. Sensitivity of the electrospray ionization is far superior to other MS ionization techniques used for nonvolatile species, (e.g., fast atom bombardment or thermospray), achieving comparable sensitivity to radioimmunoassay (RIA) methods. The proposed program will investigate various parameters that effect ES ionization (i.e. applied voltages, solvent, buffer, pH, flowrate) to achieve optimal sensitivity for opioid peptide determination. ES is a soft ionization technique, producing single and multicharged ions indictative of a peptide's molecular weight. To achieve more specificity for compound identification, the proposed program will investigate the use of ilmnobilized enzyme bioreactors and tandem MS. Immobilized enzyme bioreactors will permit hydrolysis of certain peptide bonds, specific for the particular enzyme employed. These hydrolysis products are very specific for a peptide providing information for structure confirmation. Tandem MS should provide structural fragmentation, depending on the peptide's bond strength and stability of the ion formed. The strong coulombic repulsion in multicharged lons permits the fragmentation of high molecular weight peptides by collisional activation. ES-LC/MS will be tested in a clinical situation to determine the effects of anesthesia, surgery and analgesia on the concentration of plasma pro-opiomelanocortin opioid peptides in dogs. Plasma concentration of oiioids will be evaluated by RIA and ES-LC/MS for both control dogs (anesthesia only and client dogs undergoing anesthesia and surgery. Plasma concentrations of opioids will be evaluated prior to anesthesia and up to 48 hours after extubation. Correlations between plasma opioid concentration and pain or stress-related behavior will be used to detemine the effect of analgesia therapy